这项工作由以色列科学基金会结构细胞生物学研究中心（I-CORE）（1775/12）和以色列科学基金会资助，授予1721/16和2338/16（YH）和825/14（DK ）。对"田园遗产基金会"的支持表示高度赞赏。这项工作由Ilan Edri和Michal Goldenberg完成，部分履行了特拉维夫大学萨克勒医学院的MD论文要求。

1。克隆顺式 -PT在大肠杆菌中过表达 <ol><li>获得pET-32b表达载体，设计用于与109aa硫氧还蛋白（TRX）蛋白17融合的蛋白质序列的克隆和高水平表达，以及全长顺式 -PT的大肠杆菌密码子优化的18编码序列。 </li><li>照顾到具有TEV蛋白酶（烟草蚀纹病毒蛋白酶）切割位点（ENLYFQ / G，其中，"/"表示切割点）9，随后通过短的柔性接头（SGSGSG，以增强切割位点可访问）的顺式的上游-PT序列（ 图 1A ）。另外，添加合适的5&#39;和3&#39;限制性位点进行克隆。 </li><li>克隆合成的DNA构建体导入利用标准连接技术10所述的pET-32b的载体。 </li></ol> 2。 Overexpre人DHDDS在大肠杆菌中的表达 <ol><li>转化大肠杆菌 T7根据制造商的说明书用TRX-融合DHDDS构建体表达感受态细胞。 </li><li>在氨苄青霉素选择条件（200 mg / L）下，按照制造商的说明在LB-琼脂上培养转化的细胞。 </li><li>将选择的菌落接种到250mL烧瓶中的100mL LB培养基中，培养基用100mg / L氨苄青霉素，并在37℃下恒温摇动200rpm培养过夜。 </li><li>将80mL培养物接种到两个5L烧瓶（每个烧瓶40mL）中，每个含有1.5L 2x YT培养基（16g / L胰蛋白胨，10g / L酵母提取物，5g / L NaCl），100mg / L氨苄青霉素。 </li><li>孵育培养物在37℃，以180rpm恒定振荡直到达到OD 600nm 处 = 0.5。 </li><li>将温度降至16℃，加入0.5mM异丙基β-D-1-硫代吡喃半乳糖苷（IPTG）诱导细胞。精读在相同条件下孵育16-20小时进行蛋白质表达。 </li><li>通过离心（10,000× g ，10分钟）收获细胞。 注意:协议可以暂停在这里;细胞沉淀物应保持在-80℃直至纯化。 </li></ol> 3。人类DHDDS的纯化<ol><li>将细胞重悬于缓冲液A（25mM Tris-HCl，pH7.5,150mM NaCl，0.1％Triton X-100,10mMβ-巯基乙醇），1μg/ mL DNA酶I和蛋白酶抑制剂混合物中。 </li><li>使用玻璃 - 特氟龙均质器均化细胞。 </li><li>使用微流化器或相当于12,000 - 15,000 psi 11的电池破坏电池。 </li><li>在4℃下以40,000xg离心细胞裂解物45分钟。回收含有可溶性级分的上清液。 </li><li>将上清液加载到10 - 10 mL固定化钴固定化金属亲和层析（Co 2+ -IMAC） 12柱上mM咪唑，然后用缓冲液A和10mM咪唑充分洗涤以减少非特异性蛋白质结合。 </li><li>用补充有250mM咪唑的缓冲液A洗脱过表达的蛋白质。 </li><li>用缓冲液A平衡的53 mL制备型脱盐柱除去咪唑。 </li><li>将6xHis标记的TEV蛋白酶（每50mg蛋白质1mg TEV蛋白酶）加入洗脱的蛋白质中以在4℃下将其6xHis标记的TRX融合蛋白去除过夜。 </li><li>将切割的蛋白质加载到Co 2+ -IMAC柱上，用补充有5mM咪唑的缓冲液A平衡以除去切割的6x His标记的TRX融合蛋白和TEV蛋白酶。 </li><li>收集流通并浓缩至3-4 mL，使用30 kDa分子量截留离心过滤器。 </li><li>将浓缩的蛋白质加载到用缓冲液A平衡的superdex-200大小排阻色谱柱上，以进行最终纯化。蛋白质作为二聚体（〜76 kDa）洗脱。 </LI&gt; <li>通过比较洗脱曲线与色谱柱校准曲线来选择相关级分。 </li><li>使用SDS-PAGE评估制剂的纯度。 </li><li>确定下游实验所需的蛋白质浓度和纯化的浓缩蛋白质在液氮中的闪蒸等分试样，并储存在-80°C直到使用。 </li></ol> 4。分析尺寸排阻色谱法（SEC） <ol><li>为了确保蛋白质耐受冻融循环的能力，解冻冷冻蛋白质的一部分，并以21,000 x g离心10分钟以除去不溶性聚集体。收集上清液。 </li><li>使用超高效液相色谱系统13将蛋白质加载到用25mM Tris-HCl，pH 7.5,150mM NaCl，0.02％Triton X-100,10mMβ-巯基乙醇预平衡的superdex-200分析柱上， 14 。 </li><li>监测色氨酸荧光scance（ λex = 280nm， λem = 350nm）以检测蛋白质的洗脱洗脱曲线。确保具有有效的分子量评估校准曲线 - 这些曲线可通过 SEC色谱柱制造商获得。 </li></ol> 5。酶动力学-与时间相关的活动15，16，17 <ol><li>为了确保纯化的蛋白质的活性，混合纯化的DHDDS的5μM的10μMFPP和50μM14 C-IPP以引发在缓冲液A中的反应用0.5mM的MgCl 2，在22 - 30℃。 注意:精确的反应条件可能随着不同于DHDDS的顺式 -PT的制备而变化。此外，DHDDS的活性也取决于温度和[Mg 2+ ]。 注意: 14 C-IPP是放射性的，应根据当地的辐射安全规定使用。 </LI&gt; <li>通过加入补充20mM EDTA的15μL缓冲液A（终浓度为10mM EDTA）立即骤冷反应，在0,2,4和6小时从反应中取出15μL样品。 </li><li>加入1mL H 2 O饱和的1-丁醇并充分涡旋以提取反应产物。 注意:可以在此处停止协议，并保留样品供以后读取。 </li><li>向样品中加入闪烁混合物，并使用闪烁计数器，定量包含14 C的丁醇相中的反应产物，以及反应混合物的放射性为15μL，代表总放射性。 </li><li>减去从每个时间点使用0小时样本测量的背景读数，并计算使用的14 C-IPP的百分比。 </li><li>在每个时间点计算14 C-IPP净值，计算从总共14个使用的百分比</sup&gt; C-IPP浓度，并将结果绘制为时间的函数。预期净14 C-IPP合并增加。 </li></ol>

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作者没有什么可以披露的。

这里描述的用于纯化大肠杆菌细胞中功能性人DHDDS的方案是简单和有效的，允许一旦合适的构建体可用，在3-4天内过表达和纯化蛋白质。鉴于基因组测序的突破，蛋白质纯化的这些方案具有特殊的意义，这提供了许多关于许多疾病18遗传学的信息，因此需要开发高通量方法来表征蛋白质级19的致病机制。 为了克服异源蛋白过表达和纯化中的常见缺陷，本文采取了几项措施，对成功获得可溶性和功能性蛋白质至关重要。首先，DHDDS被过度表达为TRX融合。 TRX有助于融合蛋白的折叠，增加融合蛋白的溶解性，防止包涵body形成20 。接下来，为了增加蛋白质产量，将DNA构建体密码子优化用于在大肠杆菌 21中表达。最后，为了进一步降低包涵体形成的可能性，在16℃诱导蛋白质的表达以减缓蛋白质合成速度，可能有助于蛋白质折叠22 。 考虑到不同物种中顺式 -PT的同源性，我们建议该方案可以应用于其他真核的顺式 -PT以及1 。以目前的方案为起点，给出DHDDS表达关键的一般指导方针，可以对不同顺式 -PTs的最佳表达进行修改。例如，可以尝试不同的融合配偶体（例如麦芽糖结合蛋白）或进一步优化待研究的特定蛋白质的培养条件。 &lt;P类="jove_content"&gt;随着他们的生物医学和生物技术意义1， 图7， 图8，描述的协议的将来的用途，以获得大量的纯化的官能DHDDS的，与其它顺 -PT一起，从而允许它们的结构和功能表征的追求。例如，DHDDS的进一步分子研究将解决与DHDDS相关的色素性视网膜炎的机制，潜在地导致新型治疗方法的发现和开发。此外，由于原核的顺式 -PT参与细菌壁合成，这组酶潜在地形成新的抗菌剂的新靶标。事实上，真核和原核生物酶研究的彻底表征可能允许合理开发选择性原核的顺式 -PT的抗微生物药物。最后，合成天然橡胶b顺式 -PT家族的成员，这里描述的方法可能会发现未来在天然橡胶工业合成中的应用。

异戊二烯基转移是一组催化经由多个缩合反应1使用异戊烯二磷酸（IPP）2烯丙基二磷酸，的链延长酶。 Z型酶催化从缩合反应形成顺式双键，而E型酶催化反式双键形成3 。 顺式 -磷酸转移酶（ 顺式 -PT，Z型酶）根据其产物链长度经常分类为短链（C 15 ），中链（C 50-55 ）和长链（C 70-120 ） 4 。 DHDDS（脱氢二酰基二磷酸合成酶）是一种真核长链顺式 -PT，它通过与异戊烯基二磷酸（IPP） 1的多次缩合催化法呢基二磷酸（FPP，烯丙基二磷酸）的链延长，5，6。这导致脱氢二酰基二磷酸盐的形成，C 55-100多聚丙烯酰二磷酸酯作为焦磷酸二酰基焦磷酸酯的前体，参与N-连接蛋白糖基化的糖基载体分子1 。其中德系犹太人，在DHDDS导致常染色体隐性色素性视网膜炎7,8的错义非保守突变（K42E）。因此，本方案是为了获得适用于机械研究的纯化DHDDS而开发的。 大肠杆菌被认为是重要蛋白质表达最方便和最具成本效益的宿主，因此也是最常用的宿主。然而，当试图在大肠杆菌中异源过表达蛋白质时，应该考虑蛋白质特异性的考虑。获得正确的折叠，活动来自大肠杆菌的重组蛋白质不是由于不同蛋白质的不同特性而简单的。已经开发了许多方法来克服这些障碍。在这里，蛋白质融合的使用，优化的培养条件和密码子优化被用于允许功能活跃的人DHDDS在大肠杆菌中的过表达和纯化。值得注意的是，以前尝试过度表达酵母顺式 -PT而没有蛋白质融合，因为即使在洗涤剂12的存在下也完全不溶，因此不成功。所述方案简单，经济有效，节省时间，并允许获得适合机械研究的DHDDS制剂。鉴于不同物种中顺式 -PT的同源性，我们建议该方案也可以应用于其他真核的顺式 -PT。

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			<td height="21" style="width:365px;height:21px;">pET-32b</td>
			<td style="width:100px;">Novagen</td>
			<td style="width:119px;">69016-3</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T7 Express&#160;lysY&#160;Competent&#160;E. coli&#160;(High Efficiency)</td>
			<td style="width:100px;">NEB</td>
			<td>C3010I</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:365px;height:42px;">cOmplete, EDTA-free Protease Inhibitor Cocktail</td>
			<td style="width:100px;">Roche</td>
			<td style="width:119px;">11873580001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">TALON-superflow resin</td>
			<td style="width:100px;">GE Healthcare</td>
			<td>28-9574-99</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">HiPrep 26/10 desalting column&#160;</td>
			<td style="width:100px;">GE Healthcare</td>
			<td>17508701</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">HiLoad 16/60 superdex-200&#160;</td>
			<td style="width:100px;">GE Healthcare</td>
			<td>28989335</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">superdex-200 increase 5/150 GL&#160;</td>
			<td style="width:100px;">GE Healthcare</td>
			<td>28990945</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">14C-Isopentenyl pyrophosphate</td>
			<td style="width:100px;">Perkin-Elmer</td>
			<td>NEC773050UC&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:365px;height:21px;">trans,trans-Farnesyl pyrophosphate</td>
			<td style="width:100px;">Sigma</td>
			<td>44270-10MG</td>
			<td></td>
		</tr>
	</tbody>
</table>

overexpression and purification of human cis-prenyltransferase in escherichia coli

丙二酰转移酶（PT）是一组通过多次缩合反应使用异戊烯基二磷酸酯（IPP）催化烯丙基二磷酸的链延长的酶。 DHDDS（脱氢二酰基二磷酸合成酶）是通过与异戊烯基二磷酸酯（IPP）的多次缩合催化法呢二磷酸（FPP，一种烯丙基二磷酸酯）催化链延长的真核长链顺式 -PT（从缩合反应形成顺式双键）。 DHDDS具有生物医学重要性，因为酶中的非保守突变（K42E）导致色素性视网膜炎 ，最终导致失明。因此，本协议是为了获得大量纯化的DHDDS而开发的，适用于机械研究。在这里，蛋白质融合的使用，优化的培养条件和密码子优化被用于允许功能活跃的人类DHDDS的过表达和纯化<e米&gt;电子。大肠杆菌描述的协议是简单，成本效益和时间节省。不同物种中顺式 -PT的同源性表明，该方案也可以应用于其他真核的顺式 -PT，如涉及天然橡胶合成的那些。

这里使用的构造和纯化过程的一般概述如图1所示。在每个纯化步骤获得的样品如图2所示。该SDS-PAGE分析显示DHDDS的逐步纯化，得到高度纯化的产物。 图3显示了纯化酶的分析SEC的结果，显示蛋白质仅被观察为同二聚体。 图4显示代表性的时间依赖性活性测定。 14 C-IPP掺入明显上升超过6小时，验证纯化酶是否功能。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/56430/56430fig1.jpg" /> 图1: 人DHDDS克隆和纯化 。 （ <strongA）本研究中使用的结构。 （ B ）本文所述人体DHDDS纯化方案的概要。 <a href="//ecsource.jove.com/files/ftp_upload/56430/56430fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/56430/56430fig2.jpg" /> 图2: 人DHDDS的纯化 。人DHDDS亲和纯化步骤的SDS-PAGE分析。泳道1，分子量标记（kDa）;泳道2，粗提物;泳道3，Co 2+ -IMAC流通。箭头表示TRX-DHDDS融合蛋白;泳道4，Co 2+ -IMAC洗脱液;泳道5，孵育过夜后的蛋白质-TEV蛋白酶混合物;泳道6，经TEV切割后的Co 2+ -IMAC流通;泳道7，TEV切割后的Co 2+ -IMAC洗脱液;泳道8，纯化人DHDDS（标记）由箭头所示）按照尺寸排阻色谱法。 <a href="//ecsource.jove.com/files/ftp_upload/56430/56430fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/56430/56430fig3.jpg" /> 图3: 纯化人DHDDS的分析SEC。如方案中所述监测色氨酸荧光。根据该柱的校准曲线，DHDDS形成同二聚体（77.4kDa）。箭头表示空隙体积。 <a href="//ecsource.jove.com/files/ftp_upload/56430/56430fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/56430/56430fig4.jpg" /> 图4 :Ti纯化人DHDDS的依赖于依赖的活性。 在用10μMFPP反应和50μM的14 C-IPP纯化的人DHDDS的体外活性作为底物被表示为每蛋白IPP掺入（摩尔/摩尔）。 <a href="//ecsource.jove.com/files/ftp_upload/56430/56430fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli at JoVE.com

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

来自大肠杆菌的非变性条件下用于过表达和纯化密码子优化的人顺式 -丙烯基转移酶的简单方案 大肠杆菌 ，以及酶活性测定法。该方案可以推广用于生产适合于机械研究的数量和质量的其它顺式异戊烯基转移酶蛋白质。

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

overexpression-purification-human-cis-prenyltransferase-escherichia

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

6.5K 次观看.  Tel Aviv University. 来自大肠杆菌的非变性条件下用于过表达和纯化密码子优化的人顺式 -丙烯基转移酶的简单方案 大肠杆菌 ，以及酶活性测定法。该方案可以推广用于生产适合于机械研究的数量和质量的其它顺式异戊烯基转移酶蛋白质。 

视频: Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

人外周血NK细胞的扩展，纯化和功能评估

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

科研

免疫与感染

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

在细胞命运<em&gt; E.大肠杆菌</em感染后的噬菌体λ

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes

B cells start their life with low affinity antibodies generated by V(D)J recombination. However, upon detecting a pathogen, the variable (V) region of an immunoglobulin (Ig) gene is mutated approximately 100,000-fold more than the rest of the genome through somatic hypermutation (SHM), resulting in high affinity antibodies1,2. In addition, class switch recombination (CSR) produces antibodies with different effector functions depending on the kind of immune response that is needed for a particular pathogen. Both CSR and SHM are initiated by activation-induced cytidine deaminase (AID), which deaminates cytosine residues in DNA to produce uracils. These uracils are processed by error-prone forms of repair pathways, eventually leading to mutations and recombination1-3.
Our current understanding of the molecular details of SHM and CSR come from a combination of studies in mice, primary cells, cell lines, and cell-free experiments. Mouse models remain the gold standard with genetic knockouts showing critical roles for many repair factors (e.g. Ung, Msh2, Msh6, Exo1, and polymerase &eta;)4-10. However, not all genes are amenable for knockout studies. For example, knockouts of several double-strand break repair proteins are embryonically lethal or impair B-cell development11-14. Moreover, sometimes the specific function of a protein in SHM or CSR may be masked by more global defects caused by the knockout. In addition, since experiments in mice can be lengthy, altering expression of individual genes in cell lines has become an increasingly popular first step to identifying and characterizing candidate genes15-18.
Ramos &#x2013; a Burkitt lymphoma cell line that constitutively undergoes SHM &#x2013; has been a popular cell-line model to study SHM18-24. One advantage of Ramos cells is that they have a built-in convenient semi-quantitative measure of SHM. Wild type cells express IgM and, as they pick up mutations, some of the mutations knock out IgM expression. Therefore, assaying IgM loss by fluorescence-activated cell scanning (FACS) provides a quick read-out for the level of SHM. A more quantitative measurement of SHM can be obtained by directly sequencing the antibody genes. 
Since Ramos cells are difficult to transfect, we produce stable derivatives that have increased or lowered expression of an individual gene by infecting cells with retroviral or lentiviral constructs that contain either an overexpression cassette or a short hairpin RNA (shRNA), respectively. Here, we describe how we infect Ramos cells and then use these cells to investigate the role of specific genes on SHM (Figure 1).

We describe how to perform retroviral or lentiviral infections of overexpression or shRNA-containing constructs in the human Ramos B-cell line and how to measure somatic hypermutation in these cells.

评估拉莫斯B细胞的特定基因的过表达或击倒后的体细胞超突变

B cells start their life with low affinity antibodies generated by V(D)J recombination.  However, upon detecting a pathogen, the variable (V) region of an ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac转座子初级人类T细胞系统改造

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Neuropathogenic的非侵入型<em&gt;大肠杆菌</em&gt;感染乳鼠

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. pylori-induced gastric inflammation by activating several innate leukocytes including neutrophils, monocytes, and mast cells. The immunogenic and immunomodulatory properties of HP-NAP make it a potential diagnostic and vaccine candidate for H. pylori and a new drug candidate for cancer therapy. In order to obtain substantial quantities of purified HP-NAP used for its clinical applications, an efficient method to purify this protein with high yield and purity needs to be established.
In this protocol, we have described a method for one-step negative chromatographic purification of recombinant HP-NAP overexpressed in Escherichia coli (E. coli) by using diethylaminoethyl (DEAE) ion-exchange resins (e.g., Sephadex) in batch mode. Recombinant HP-NAP constitutes nearly 70% of the total protein in E. coli and is almost fully recovered in the soluble fraction upon cell lysis at pH 9.0. Under the optimal condition at pH 8.0, the majority of HP-NAP is recovered in the unbound fraction while the endogenous proteins from E. coli are efficiently removed by the resin.
This purification method using negative mode batch chromatography with DEAE ion-exchange resins yields functional HP-NAP from E. coli in its native form with high yield and purity. The purified HP-NAP could be further utilized for the prevention, treatment, and prognosis of H. pylori-associated diseases as well as cancer therapy.

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

A high yield method for one-step negative purification of recombinant Helicobacter pylori neutrophil-activating protein (HP-NAP) overexpressed in Escherichia coli by using diethylaminoethyl resins in batch mode is described. HP-NAP purified by this method is beneficial for the development of vaccines, drugs, or diagnostics for H. pylori-associated diseases.

一步法负色谱纯化<em&gt;幽门螺杆菌</em&gt;中性粒细胞激活蛋白过度表达<em&gt;大肠杆菌</em&gt;在批处理模式

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

非侵入性 体内生物发光系统检测小鼠宿主肠出血性大肠杆菌的定植

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

使用细菌共轭的 Enterobacterial 菌株 (如大肠杆菌或志贺菌福氏) 创建稠密的转插入库

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. Among them, reversible lysine acetylation is one of the most widely distributed in all domains of life. Although numerous mass spectrometry-based acetylome studies have been performed, further characterization of these putative acetylation targets has been limited. One possible reason is that it is difficult to generate purely acetylated proteins at desired positions by most classic biochemical approaches. To overcome this challenge, the genetic code expansion technique has been applied to use the pair of an engineered pyrrolysyl-tRNA synthetase variant, and its cognate tRNA from Methanosarcinaceae species, to direct the cotranslational incorporation of acetyllysine at the specific site in the protein of interest. After first application in the study of histone acetylation, this approach has facilitated acetylation studies on a variety of proteins. In this work, we demonstrated a facile protocol to produce site-specifically acetylated proteins by using the model bacterium Escherichia coli as the host. Malate dehydrogenase was used as a demonstration example in this work.

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Genetic code expansion serves as a powerful tool to study a wide range of biological processes, including protein acetylation. Here we demonstrate a facile protocol to exploit this technique for generating homogeneously acetylated proteins at specific sites in Escherichia coli cells.

在大肠杆菌中生成特异的乙酰蛋白的简易协议

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

人类的过表达和纯化

摘要

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此视频中的章节

人外周血NK细胞的扩展，纯化和功能评估

在细胞命运

评估拉莫斯B细胞的特定基因的过表达或击倒后的体细胞超突变

piggyBac转座子初级人类T细胞系统改造

Neuropathogenic的非侵入型

一步法负色谱纯化

人全血中NK细胞杀伤活性的流式细胞仪分析

非侵入性体内生物发光系统检测小鼠宿主肠出血性大肠杆菌的定植

使用细菌共轭的 Enterobacterial 菌株 (如大肠杆菌或志贺菌福氏) 创建稠密的转插入库

在大肠杆菌中生成特异的乙酰蛋白的简易协议

人类的过表达和纯化

摘要

探索更多视频

此视频中的章节

人外周血NK细胞的扩展，纯化和功能评估

在细胞命运

评估拉莫斯B细胞的特定基因的过表达或击倒后的体细胞超突变

piggyBac转座子初级人类T细胞系统改造

Neuropathogenic的非侵入型

一步法负色谱纯化

人全血中NK细胞杀伤活性的流式细胞仪分析

非侵入性 体内生物发光系统检测小鼠宿主肠出血性大肠杆菌的定植

使用细菌共轭的 Enterobacterial 菌株 (如大肠杆菌或志贺菌福氏) 创建稠密的转插入库

在大肠杆菌中生成特异的乙酰蛋白的简易协议

非侵入性体内生物发光系统检测小鼠宿主肠出血性大肠杆菌的定植